Biomedical subjects

Wojciech P Pawlowski

Publications and source records attributed to Wojciech P Pawlowski.

5 recordsLinked to original sources

PubMed2026

Detecting Meiotic Crossing-Overs in Maize Using Chromatin Immunoprecipitation-Sequencing (ChIP-seq).

During meiosis, homologous chromosomes engage in reciprocal exchanges of segments in a process known as crossing over (CO). About 85% of CO events in maize are products of the class I pathway. Class I COs are interference-sensitive, meaning that the formation of one CO reduces the likelihood of another CO forming close by. This protocol describes a chromatin immunoprecipitation-sequencing (ChIP-seq)-based method for mapping meiotic COs in maize, using an antibody against MutL Homolog 3 (MLH3), a key component of the class I CO pathway. CO sites are determined by Illumina sequencing of DNA isolated from MLH3-associated chromatin fragments. Traditionally, COs have been identified through genetic mapping, which relies on the segregation of genetic markers in the progeny of hybrid plants. However, conventional genetic mapping provides limited resolution and requires large numbers of progeny individuals. The MLH3 ChIP-seq approach enables direct detection of COs, providing high-resolution and genome-wide coverage, including genome regions with low DNA sequence polymorphism, which are inaccessible to genetic CO mapping. Furthermore, MLH3 ChIP-seq enables screening of thousands of CO events, greatly accelerating the analysis and reducing its cost. This protocol can also be used to examine any chromatin-bound meiotic proteins and adapted to studying chromatin-associated proteins in somatic cells.

Journal Article↗

PubMed2026

Cytological Assessment of Maize Pollen Viability Using a Simplified Staining Protocol.

Anomalies of meiosis frequently result in abnormal chromosome segregation, which leads to defects in pollen formation in maize. Thus, assessing pollen viability is an important measure for examining the overall success of male sexual reproduction. Pollen viability tests are used to characterize mutants defective in meiosis and microsporogenesis, and to determine the effects of genome instability and environmental conditions on reproduction. This protocol describes a rapid method for assessing pollen viability in maize, using a simplified cytological staining approach. Traditional pollen staining methods, such as Alexander staining, often require hazardous chemicals that are increasingly restricted due to safety concerns. The method presented here uses easily accessible reagents and avoids highly toxic substances.

Journal Article↗

PubMed2026

Site-Specific Measurement of Meiotic Crossing-Over Rate with Droplet Digital PCR.

Understanding the frequency and distribution of meiotic crossovers (COs) is critical for both fundamental studies on meiosis and for practical applications in plant breeding, where controlling recombination can accelerate crop improvement. Determining CO rates at specific genomic loci has traditionally relied on labor-intensive methods that require the production and genotyping of large progenies. Here, we present a high-throughput protocol for site-specific quantification of meiotic COs in maize using droplet digital PCR (ddPCR). The method is based on genotyping individual pollen nuclei from hybrid plants to detect recombinant and nonrecombinant alleles at defined chromosomal intervals. By distributing several thousands of pollen nuclei into nanoliter-sized droplets and performing PCR with allele-specific fluorescent probes, this method allows precise quantification of CO frequency with high sensitivity. The protocol provides detailed guidance for nuclei isolation, probe master mix preparation, droplet generation, and data interpretation. This method can be easily adapted for use in other plants.

Journal Article↗

PubMed2026

Analyzing Meiosis in Maize.

Meiosis is central to sexual reproduction and the main source of genetic diversity in plants. Understanding how meiotic processes are regulated has direct relevance to agriculture. As meiotic recombination is the vehicle of plant breeding, gaining the ability to influence recombination patterns can accelerate crop improvement. Maize is a powerful model for studying plant meiosis, thanks to its large chromosomes, well-developed genetics, and the availability of diverse cytogenetic and molecular tools. Insights gained from maize studies can extend to other species. In this review, we describe a variety of approaches for examining meiosis and meiotic recombination in maize. Cytological techniques, including protein immunolocalization and fluorescence in situ hybridization (FISH), enable visualization of chromosome structure and behavior, as well as crossover (CO) formation. Chromatin immunoprecipitation (ChIP) is used in meiosis research to determine locations of recombination proteins, identify recombination sites, and elucidate chromatin features, such as histone modifications. Quantification of COs at specific genomic sites through pollen typing by droplet digital PCR allows precise high-resolution measurement of recombination rates. Combining cytology, protein localization, and molecular assays provides a multiscale picture of meiosis, linking molecular mechanisms to chromosome behavior and, ultimately, to genetic variation.

Journal Article↗

PubMed2026

Evolution of maize recombination landscape during domestication.

Despite the plethora of knowledge about the benefits of meiotic recombination and numerous theoretical studies examining how recombination rates evolve, there is a general lack of empirical support and consensus across species. To fill this knowledge gap, we characterized the evolution of recombination landscape in maize during its domestication from teosinte and related the observed changes to established theoretical frameworks. Through examining recombination in experimental populations of maize and teosinte and the population genomics approach of identifying historical recombination events using ancestral recombination graph inference to generate saturated maize and teosinte recombination maps, we found that during domestication, maize experienced a 12% increase in its genome-wide recombination rate. Furthermore, maize evolved higher recombination rates on the long arms of chromosomes in regions closer to centromeres, where recombination is generally very low. The repatterning of crossover events came from changes in global crossover positioning rather than alterations in cis-acting chromatin factors. Consequently, we found evidence of selection acting on trans-acting recombination modifiers affecting crossover interference and controlling the interference-dependent class I crossover pathway. We show that CO repatterning was likely beneficial for maize fitness, as significant recombination rate increases were predominantly in gene-rich regions, which harbor domestication-related variation. This work suggests genomic and mechanistic processes leading to the evolution of meiotic recombination landscape in response to directional selection pressure and provides evidence for the evolutionary advantage of recombination.

Zea mays↗